RNA干扰细胞周期蛋白依赖激酶6基因对尤文肉瘤细胞生物学行为的影响

背景与目的：细胞周期蛋白依赖激酶6(cyclin-dependent kinase 6，CDK6)是一类丝/苏氨酸蛋白激酶，有实验报道其在尤文肉瘤细胞中呈高表达，但在尤文肉瘤细胞中的生物学功能及相关机理仍不十分清楚。本研究旨在通过研究小干扰RNA(small interfering RNA，siRNA)沉默CDK6基因表达，探讨其对尤文肉瘤A673、SK-ES-1细胞生物学行为的影响。方法：将化学合成的CDK6-siRNA转染至A673及SK-ES-1细胞中，实时荧光定量PCR法检测转染前后细胞中CDK6 mRNA的表达；蛋白质印迹法(Western blot)检测转染前后细胞CDK6以及CDK6下游因子视网膜母细胞瘤基因(Rb)、磷酸化Rb(P-Rb)的蛋白表达；分别采用CCK-8法、流式细胞仪、Transwell肿瘤细胞迁移和侵袭实验检测细胞增殖、周期、凋亡、迁移以及侵袭能力的变化。结果：实时荧光定量PCR结果显示，CDK6-siRNA能够明显降低细胞CDK6的表达(P<0.01)。抑制CDK6表达后细胞的增殖速度明显减缓，细胞周期分布发生改变，G0/G1期细胞的比例上升，而S期细胞的比例下降，细胞的早期凋亡率明显升高(P<0.01)。各组细胞的迁移能力：untreated组、si-con组及si-CDK6组A673细胞的穿膜细胞数分别为(516.00±58.59)个、(534.00±124.77)个、(192.33±68.92)个，SK-ES-1细胞的穿膜数分别为(371.33±29.67)个、(363.33±60.28)个、(200.00±20.00)个，差异有统计学意义(P<0.01)；各组细胞的侵袭能力：untreated组、si-con组及si-CDK6组A673细胞的穿膜细胞数分别为(251.00±42.93)个、(238.67±78.62)个、(94.67±23.03)个，SK-ES-1细胞的穿膜数分别为(310.00±35.36)个、(302.33±41.31)个、(105.00±54.08)个，差异有统计学意义(P<0.01)。Western blot检测结果显示，CDK6-siRNA能够明显降低细胞CDK6以及下游因子P-Rb的表达(P<0.01)，但细胞总Rb表达量无明显变化。结论：针对CDK6基因的特异性小RNA干扰片段能够下调CDK6基因及蛋白的表达，并抑制尤文肉瘤细胞增殖、侵袭和迁移，诱导其凋亡。

Effects of a small interfering RNA targeting CDK6 gene on the biological behaviors of Ewing’s sarcoma cells

Background and purpose: Cyclin-dependent kinase 6 (CDK6) is a kind of serine/threonine protein kinase, which has been reported to be over-expressed in the Ewing’s sarcoma cells. However, the biological effects and relative mechanisms of CDK6 on Ewing’s sarcoma cells are really less known. This study aimed to investigate the effects of CDK6 gene on the biological behaviors of Ewing’s carcinoma cells A673 and SK-ES-1, we knocked down CDK6 using a small interfering RNA targeting CDK6. Methods: The A673 and SK-ES-1 cells were transfected with a chemical synthesized small interfering RNA which targets CDK6; The real time-PCR and Western blot assays were performed to detected the mRNA and protein expression levels of CDK6 and its downstream genes Rb and P-Rb in both cells transfected with the siRNAs; The CCK-8, flow cytometry (FCM), migration and invasion assays were performed to indentify the proliferation, cell cycle distribution, apoptosis, migration and invasion abilities after the transfection with si-CDK6, respectively. Results: The real time-PCR analysis showed the expression of CDK6 was down-regulated in the A673 and SK-ES-1 cells transfected with si-CDK6 (P<0.01); Suppression of CDK6 by si-CDK6 inhibited the cell proliferation, induced cell apoptosis andG₀/G₁-Phase cell cycle arrest (P<0.01); The cells of A673 migrating through the membrane in untreated group, si-con group and si-CDK6 group were 516.00±58.59, 534.00±124.77 and 192.33±68.92, respectively. The numbers of SK-ES-1 cells were 371.33±29.67, 363.33±60.28 and 200.00±20.00, respectively. The results showed significant differences in statistics (P<0.01); In the invasion assay, the cells of A673 passed through the Matrigel coated membrane in untreated group, si-con group and si-CDK6 group were 251.00±42.93, 238.67±78.62 and 94.67±23.03, respectively, and the number of SK-ES-1 cells were 310.00±35.36, 302.33±41.31 and 105.00±54.08, respectively. The results showed down-regulation of CDK6 could suppress the cells migration and invasion abilities (P<0.01); The Western blot analysis showed the protein levels of CDK6 and its downstream gene phospho-retinoblastoma gene (P-Rb) were down-regulated, while there were no changes in the expression of Rb after transfection.Conclusion: CDK6 siRNA specifically and efficiently blocks the constitutively activated CDK6 in human Ewing’s sarcoma cells, resulting in inhibition in cell proliferation, migration, invasion and induction of cell apoptosis.